Alcohol consumption is the world?s third largest risk factor for disability and disease. Binge alcohol drinking has been linked to many adverse social and health consequences, including an increased risk of transitioning to alcohol dependence. Stress is a key environmental factor in alcohol use and has been linked to alcohol use disorders (AUDs) in clinical and preclinical models. The key CNS regulator of stress is corticotropin-releasing factor (CRF), and CRF plays an important role in modulating binge drinking (BD) and dependence. The CRF system includes two distinct GPCRs, CRF receptor 1 (CRF-R1) and CRF receptor 2 (CRF-R2), and a secreted CRF-binding protein (CRF-BP) that binds CRF with an affinity equal to or greater than the receptors. While CRF and CRF-R1 have been clearly implicated in AUDs, CRF-R1 antagonists have been ineffective in clinical trials. Strikingly, CRHBP SNPs have been associated with BD and dependence, CRF-BP expression is regulated in BD, and rodent pre-clinical studies suggest that blocking CRF-BP activity in VTA may decrease BD. Yet, the functional roles of CRF-BP within the stress/reward pathways and its modulation of CRF receptor activity in vivo are not well understood. Dissecting the specific roles of CRF-BP in different brain regions in binge and heavy alcohol drinking may enable the discovery of new therapeutic treatments to reduce BD and prevent escalation to dependence. Secreted CRF-BP inhibits CRH activity at CRF-R1, while CRF-BP appears to enhance CRF-R2 signaling in VTA; other studies suggest that intracellular CRF-BP acts as an escort protein for CRF-R2. We propose that CRF-BP plays multiple roles, likely in a brain region or cell-type specific manner, with CRF receptor subtype as a potential determinant. In this R21 proposal, we will focus on 2 key interacting sites of CRF-BP/CRF receptor co-expression within the stress/reward pathways, the prefrontal cortex (mPFC) and ventral tegmental area (VTA). Using a combination of pharmacological (CRF6-33 and receptor antagonists), viral, and genetic tools, we will compare the intracellular and extracellular roles of the CRF-BP in the cell types normally expressing CRF-BP in the mPFC and VTA. We will utilize the drinking in the dark (DID) and chronic intermittent access to alcohol (IAA) models to assess the role of CRF-BP across the transition from BD to dependence-like drinking. Both male and female mice will be studied as sex-specific changes in CRF and CRF-R2 expression have recently been reported in BD. We will examine the role of CRF-BP in mPFC (Aim 1) and VTA (Aim 2) using an extracellular CRF-BP antagonist (CRF6-33), viral CRF-BP knockdown, and cre- dependent overexpression of CRF-BP in mPFC sst-expressing cells (Aim 1C) and VTA TH-expressing neurons (Aim 2C). We hypothesize that CRF-BP has largely inhibitory roles at CRF-R1 in PFC, while CRF-BP in VTA may have both inhibitory and enhancing roles via CRF-R1 and CRF-R2 respectively. Results from this project will advance our understanding of brain region and cell-specific roles of CRF-BP, as well as characterize the potential for CRF-BP antagonists as a novel therapeutic target for treatment of AUDs.